TGR5 agonists induce peripheral and central hypersensitivity to bladder distension

The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladder-innervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1−/− but not Trpa1−/− DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3β-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1−/− mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.


Methods
Ex vivo bladder afferent nerve recordings. Nerve recordings were performed in an ex vivo model as previously described 16,17,[35][36][37] . Mice were humanely killed via CO 2 inhalation, and the entire lower abdomen was removed and submerged in a modified organ bath under continual perfusion with gassed (95% O 2 and 5% CO 2 ) Krebs-bicarbonate solution (composition in mmol/L: 118.4 NaCl, 24.9 NaHCO 3 , 1.9 CaCl 2 , 1.2 MgSO 4 , 4.7 KCl, 1.2 KH 2 PO 4 , 11.7 glucose) at 35 °C. The bladder, urethra, and ureters were exposed by removing excess tissue. Ureters were tied with 4-0 perma-hand silk (Ethicon, #LA53G). The bladder was catheterised (PE 50 tubing) via the urethra and connected to a syringe pump (NE-1000) to allow a controlled fill rate of 30 µL/min with saline (NaCl, 0.9%). To enable intravesical pressure recording during graded distension, a second catheter was inserted through the dome of the bladder, secured with silk, and connected to a pressure transducer (NL108T2; Digitimer). Pelvic nerves, isolated from all other nerve fibres between the pelvic ganglia and the spinal cord, were dissected into fine multiunit branches and a single branch was placed within a sealed glass pipette containing a microelectrode (WPI) attached to a Neurolog headstage (NL100AK; Digitimer). Nerve activity was amplified (NL104), filtered (NL 125/126, band pass 50-5000 Hz, Neurolog; Digitimer), and digitised (CED 1401; Cambridge Electronic Design, Cambridge, UK) to a PC for offline analysis using Spike2 software (Cambridge Electronic Design, Cambridge, UK). The number of action potentials crossing a pre-set threshold at twice the background electrical noise was determined per second to quantify the afferent response. Single-unit analysis was performed offline using Spike2 software by matching individual spike waveforms through linear interpolation. A single unit was deemed to be responsive if a greater than 20% increase in afferent responsiveness was detected. Afferent recording experimental protocols. As described previously 16,17,[35][36][37] , at the start of each afferent recording experiment, control bladder distensions were performed with an intravesical infusion of saline (NaCl, 0.9%) at a rate of 30 µL/min to a maximum pressure of 30 mmHg at 10 min intervals to assess the viability of the preparation and reproducibility of the intravesical pressure and neuronal responses to distension. The volume in the bladder was extrapolated from the known fill rate (30 µL/min) and the time taken (s) to reach maximum pressure (30 mmHg). Compliance was determined by plotting intravesical pressure against the calculated volume. After a stable baseline was maintained, the saline in the infusion pump was replaced by 5α-pregnan-3β-ol-20-one sulphate (Pg5α; 300 µM; Steraloids, #P3865-000) or 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5dimethyl-4-isoxazolecarboxamide (CCDC; 300 µM; Biovision, #1722-5, CAS: 1197300- . Pg5α is a progesterone sulphate previously identified as an endogenous TGR5 agonist 28 , whilst CCDC is a potent synthetic TGR5 agonist reported to be TGR5-specific 38 . For both Pg5 α and CCDC, stock solutions were made to 10 mM with 1% DMSO. Stock solutions were diluted to final working concentrations in 0.9% saline. Control refers to 0.9% saline containing 0.03% DMSO. The single nerve bundle isolated and inserted into the glass electrode during the dissection process was contained within the recording electrode for the entire experiment, allowing afferent nerve responses to be compared in the same nerve fibres during intra-bladder incubation with saline and Pg5α (300 µM) or CCDC (300 µM).
In vivo spinal dorsal horn activation. In vivo bladder distension. In vivo bladder infusion in anaesthetised mice were performed as previously described 16,37 . 12-week-old female mice were anaesthetised (isoflurane 2-4% in oxygen) and a catheter (PE 50 tubing) was inserted into the bladder via the urethra. Correct catheter placement was determined by inserting the catheter gently until meeting resistance (bladder dome) and receding slightly (2-3 mm) to avoid damaging the bladder wall. Urine was removed using a suction syringe and the Scientific Reports | (2022) 12:9920 | https://doi.org/10.1038/s41598-022-14195-w www.nature.com/scientificreports/ catheter removed. A new catheter (PE 50 tubing), primed with either vehicle (saline; N = 5 mice) or CCDC (100 µM; N = 5 mice), was inserted and 100 µL of solution infused gently, to fill but not fully distend the bladder, and allowed to incubate for 5 min. The solution was then carefully removed from the bladder to ensure minimum liquid remained in the bladder. A third, dry catheter (PE 50 tubing) was inserted, and a small length of 4-0 perma-hand silk (Ethicon, #LA53G) was knotted over the urethral opening to fix the catheter into place and minimise loss of pressure. A 5-min stabilisation period was allowed between tying the catheter and performing the distensions to limit false-positive pERK signalling from the act of tying. A sphygmomanometer was attached between the catheter and a 10 mL air-filled suction syringe. 5 × 20-s bladder distensions were performed at 10-s intervals, at a holding pressure of 40 mmHg. Following the fifth 20-s distension, mice were administered an euthanasia agent overdose (intraperitoneal injection, 0.125 mL/250 g Lethabarb ® ; Virbac Australia) followed by rapid transcardial perfuse fixation.
Transcardial perfuse fixation and tissue dissection. As described previously 16,17,[35][36][37] , following euthanasia overdose, the thoracic cavity was opened and 0.5 mL heparinised saline (50 IU  The frozen spinal cord segments were cryosectioned (10 µm thickness) and placed onto gelatin-coated slides for processing of immunofluorescence labelling. Sectioning was performed serially and distributed over 6 slides, which were used for immunofluorescence localisation of phosphorylated-MAP-kinase ERK 1/2 (pERK). The number of pERK-immunoreactive neurons were counted in spinal cord sections from L6-S1, with spinal segments determined ex vivo by the shape of the dorsal horn 39 .
Immunohistochemistry of phosphorylated-MAP-kinase ERK 1/2 within the spinal cord. The dorsal horn neurons activated by bladder distension were identified by immunofluorescent labelling for neuronal activation marker, phosphorylated-MAP-kinase ERK 1/2 (pERK), using a method described previously 16,36,37,40,41 . The details for the primary and secondary antisera can be found in Table 1. pERK activation was determined rather than c-Fos as ERK phosphorylation occurs within minutes of the peripheral stimuli and thus correlates better to the shortduration peripheral stimuli applied. Further, spinal neuronal activation in response to a range of peripheral stimuli is increasingly being identified via pERK activation quantification 36,37,41,42 . After air drying for one hour, excess OCT was removed from sections by washing with 0.2% Triton X-100 (Sigma-Aldrich, #T8787) in 0.1 M phosphate-buffered saline (PBS-T). Non-specific secondary antibody binding was blocked with 5% normal chicken serum diluted in 0.2% PBS-T. Tissue sections were then incubated with pERK primary antisera diluted in PBS-T at room temperature overnight (18 h). Sections were washed in PBS-T and incubated with secondary antibody conjugated to AlexaFluor 594 for 1 h at room temperature, followed by a PBS-T wash before mounting in ProLong Diamond Antifade (Life Technologies, #P-36962) and coverslipping. Negative controls were prepared as above with omission of the primary antibody. Slides were allowed to set for 24 h prior to visualisation.
Microscopy. Fluorescence was visualised with an epifluorescence microscope (Olympus, Tokyo, Japan) using a 10 × objective and 3-s exposure. ImageJ software (NIH) was used for image analysis. Aside from making moderate adjustments for brightness and contrast, the images were not manipulated in any way.
Spinal cord pERK neuronal counts and analysis. Neuronal counts were analysed as described previously 16 and utilized previously saved digital photomicrographs, with only neurons with intact nuclei counted. The number of pERK-immunoreactive (pERK-IR) neurons in a quadrant of the L6-S1 dorsal horn was obtained from a minimum of 6 sections/animal viewed at 10 × magnification. The mean number of pERK-IR neurons (± SEM) was compared between vehicle-and CCDC-treated mice.  36,37 . The needle was inserted subserosally, parallel with the bladder muscle, to prevent injection of CTB into the bladder lumen. The abdominal incision was sutured closed and antibiotic (Amoxicillin; 50 mg/kg; Amoxil, AUSTR11137) and analgesic (Buprenorphine (Temvet); 0.1 mg/kg; Troy Laboratories Pty Ltd, APVMA #67612) given subcutaneously as mice regained consciousness. Mice were then individually housed and allowed to recover. 4 days postsurgery, mice were humanely killed for subsequent removal of the lumbosacral (LS; L5-S1) dorsal root ganglia (DRG) and isolation and culture of the neurons to visualise CTB-labelled bladder-innervating neurons within the DRG population.
Cell culture of bladder-innervating DRG neurons. As described previously 16

Single-cell RT-PCR of individual bladder-innervating dorsal root ganglia neurons.
Under continuous perfusion of sterile and RNA-/DNase-free PBS, single retrogradely traced bladder dorsal root ganglia (DRG) neurons were identified using a fluorescence microscope and collected into the end of a fine glass capillary using a micromanipulator 16,37 . The glass capillary was then broken into a sterile Eppendorf tube containing 10 µL of lysis buffer with DNAse (TaqMan Gene Expression Cells-to-CT Kit; Invitrogen, #4399002). For each coverslip of cultured DRG neurons, a bath control was also taken and analysed together with cells. Following lysis and termination of DNAse treatment, samples were immediately frozen on dry ice and stored at − 80 °C until cDNA synthesis was performed. RNA was reverse transcribed to cDNA using SuperScript™ IV VILO™ Master Mix (Invitrogen, #11766500) as per the manufacturer's instructions. cDNA was stored at − 20 °C for qRT-PCR. Tubb3 (tubulin-3) expression served as a neuronal marker and positive control, and Gfap (glial fibrillary acidic protein) expression served as a glial cell marker for exclusion. Expression was considered positive if a complete curve was observed before 50 cycles 16 .
Analysis of mRNA expression in whole LS DRG, isolated urothelial cells and bladder mucosal and detrusor layers. Tissue collection. As described previously 16 16-to 18-week-old mice were humanely euthanised via CO 2 inhalation and the bladder and lumbosacral (L5-S1) dorsal root ganglia (DRG) removed. DRG were frozen in dry ice in pairs by spinal level (L5, L6, S1) and stored at -80 °C for RNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The bladder was dissected in sterile PBS with a single cut from the urethral opening along one side to the dome, stretched out, and pinned flat, urothelial side up. For RT-qPCR of the bladder layers, the mucosa and detrusor were gently peeled apart, frozen in dry ice, and stored at -80˚C for RNA extraction and qRT-PCR.

Isolation of mouse urothelial cells.
For urothelial cell qRT-PCR, urothelial cells were isolated as performed previously 16,35,44,45 . Following stretching and pinning of the opened bladder as per bladder layer separation, the bladder was incubated with DMEM containing 2.5 mg/mL dispase, 10% Foetal Calf Serum, and 1 µM/mL HEPES (Sigma, #H3375, pH 7.0) at room temperature for 3 h. Cells were collected by gentle scraping of the urothelium with a blunt scalpel and dissociated in 0.025% trypsin EDTA (GIBCO, ThermoFisher Scientific, #25200072) at 37 °C in 5% CO 2 for 15 min with gentle intermittent trituration 16 . The trypsin was deactivated by adding the cell suspension to DMEM containing 10% Foetal Calf Serum before centrifugation (15 min, 1500 rpm, 4 °C). Cells were then resuspended in keratinocyte serum-free media (KSFM; Invitrogen, #17005042). Cell count and viability were determined using the Countess Automated Cell Counter (Invitrogen, ThermoFisher Scientific). Cells were then pelleted, frozen in dry ice, and stored at − 80 °C for RNA extraction and qRT-PCR.
RNA extraction and cDNA synthesis. As described previously 16  Fluorescence images were obtained every 5 s leading up to and immediately following drug application, and every 20 s for washout period, using a 20× objective. Data were recorded and further analysed using MetaFluor software.
After an initial baseline reading to ensure cell fluorescence was stable, indicating healthy cells, DRG neurons were stimulated with CCDC (100 µM; Biovision, #1722-5, CAS: 1197300-24-5), and changes in intracellular calcium [Ca 2+ ] i were monitored in real time. For experiments assessing downstream TRP channel signalling, application and washout of CCDC was followed by application of TRPA1 agonist allyl isothiocyanate (AITC; 100 µM; Aldrich, #377430, CAS: 57-06-7) then TRPV1 agonist Capsaicin (50 nM; Sigma, #M2028, CAS: 404-86-4). After agonist additions, HEPES buffer containing high KCL (40 mM) was applied as a positive control to induce calcium overload driven by neuronal depolarisation. [Ca 2+ ] i is expressed as the ratio between the fluorescence signals at 340 and 380 nm. Baseline [Ca 2+ ] i was determined by averaging 8-12 fluorescence ratio readings immediately prior to stimulation. Maximum peak response was calculated by subtracting the relevant baseline score from the maximum fluorescence ratio measurement following compound application. Baseline fluorescence was normalised to a ratio of 1 for example traces.
Data availability, experimental design, data analysis and statistics. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Data are presented as described previously 16 with the mean ± SEM or the % of neurons or afferents or the maximum imp/s before and after. N indicates the number of animals, whilst n indicates the number of independent neurons or afferents. Statistical significance was reported at a level of *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. In some cases, # is used to indicate significance of at least P < 0.05 for multiple comparisons. Data were analysed using Prism 8 (GraphPad, San Diego, CA, USA), using one-or two-way analysis of variance (ANOVA) with Tukey's or Sidak's post hoc analyses dependent on data distribution, or student t-tests, for parametric data. Nonparametric data were analysed using Kruskal-Wallis test with Dunn's multiple comparisons, Mann-Whitney Table 2. Primers used for qRT-PCR receptor expression assays.

Ethics and animals.
Animal studies were performed and reported in compliance with the ARRIVE guidelines. In this study, we used 12-to 18-week-old male and female C57BL/6J mice provided via an in-house breeding colony at the South Australian Health and Medical Research Institute (SAHMRI). Mice at SAHMRI were originally acquired from an in-house C57BL/6J breeding programme (JAX strain #000664; originally purchased from The Jackson Laboratory; breeding barn MP14; Bar Harbor, ME) and then bred within the specific and opportunistic pathogen-free animal care facility at SAHMRI. Some experiments utilised TRPV1 knockout (trpv1 −/− ), TRPA1 knockout (trpa1 −/− ), or TGR5 knockout (Gpbar1 −/− ) mouse strains with a C57BL/6J genetic background, also bred in-house at SAHMRI. Trpv1 −/− mice were originally acquired from an in-house breeding program at The Jackson Laboratory (JAX stock #003770; Bar Harbor, ME), Trpa1 −/− mice were gifted to us by the original developer, Prof David Corey (Harvard University, Cambridge, MA, USA), and Gpbar1 −/− mice were gifted to use by Prof Johan Auwerx and Prof Kristina Schoonjans, Ecole Polytechnique de Lausanne (Lausanne, Switzerland). Gpbar1 −/− mice were also crossed onto Na V 1.8cre-line to generate a sensory neuron specific These genetically modified mouse lines were all then bred in-house at SAHMRI as per the wildtype mice. Na V 1.8Cre-Gpbar1 −/− mice were gifted to us by Prof Nigel Bunnett (Monash University, Victoria, Australia), then housed at SAHMRI's Preclinical, Imaging, and Research Laboratories (PIRL) facility for procedures. All experiments were approved by and performed in accordance with the SAHMRI Animal Ethics Committee approvals (SAM195 and SAM190). Mice were group housed (5 mice per cage) within individual ventilated cages filled with chip coarse dust-free aspen bedding (PuraChips Aspen coarse 63L; Cat# ASPJMAEB-CA, Able Scientific, Australia). Cages were stored on individual ventilated cage racks within a temperature-controlled environment of 22 °C and a 12-h light/12-h dark cycle. Mice had free access to LabDiet JL Rat and Mouse/Auto6F chow (Speciality Feeds, Australia) and autoclaved reverse osmosis water. Sample sizes of each experiment are detailed in the corresponding figure legends.

Results
The TGR5 agonist Pg5α causes bladder afferent hypersensitivity. To determine if activation of the TGR5 receptor could influence bladder sensation we determined the impact of peripheral TGR5 activation in an ex vivo bladder afferent preparation. Intravesical application of Pg5α, a progesterone sulphate previously identified as an endogenous TGR5 agonist 28 , resulted in a significant increase in bladder afferent firing in response to graded bladder distension compared to saline (Fig. 1A), indicating mechanical hypersensitivity following TGR5 activation. No change was observed in bladder compliance (pressure/volume relationship) between saline and Pg5α experiments (Fig. 1B), indicating that afferent hypersensitivity was not caused by changes in detrusor muscle tone.
Single-unit analysis of the bladder afferent nerve fibres revealed that 44.4% (20/45) of the mechanosensitive bladder afferents demonstrated mechanical hypersensitivity following instillation of Pg5α ('responders'), whereas 55.6% (25/45) of mechanosensitive afferents were unresponsive to Pg5α ('non-responders') ( Fig. 1C). Pg5α-responsive afferents showed a significantly increased afferent firing rate at pressures of 20 mmHg and above (Fig. 1Di) and a significant increase in the maximum peak of afferent firing in response to distension following Pg5α instillation (Fig. 1Dii). In comparison, no change in afferent firing was observed between saline and Pg5α experiments for non-responding afferents, neither at any particular distension pressure (Fig. 1Ei) nor in the maximum peak response (Fig. 1Eii). These data demonstrate that the progesterone metabolite Pg5α causes mechanical hypersensitivity of a sub-population of bladder afferents in response to graded bladder distension (Fig. 1F).
The synthetic TGR5 agonist CCDC replicates Pg5α-induced increases in bladder afferent mechanosensitivity. We next aimed to further define and confirm the specific involvement of the TGR5 receptor in mediating bladder afferent mechanosensitivity. To this end, we used a potent, synthetic TGR5 agonist, 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethyl-4-isoxazolecarboxamide (CCDC), reported to be TGR5specific 38 to confirm the results obtained with Pg5α. Similar to Pg5α, on applying CCDC to the ex vivo bladder afferent preparation we observed an increase in afferent mechanosensitivity to bladder distension compared to saline ( Fig. 2A). No change to muscle compliance was observed between saline and CCDC intravesical instillation (Fig. 2B), demonstrating that the effect of CCDC on mechanosensitivity is a direct afferent effect and not secondary to muscular changes. Single unit analysis of the mechanosensitive bladder afferents showed 50% (20/40) of afferents responded to CCDC with mechanical hypersensitivity to bladder distension compared to saline ('responders'), while 50% (20/40) showed no change in mechanosensitivity following CCDC instillation ('non-responders') compared to saline (Fig. 2C). CCDC-responsiveness was indicated by a mechanical hypersensitivity compared to saline at pressures of 10 mmHg and above (Fig. 2Di) and by a significant increase in the maximum peak of afferent firing (Fig. 2Dii) following CCDC instillation compared to saline. No changes in afferent firing at any individual pressures (Fig. 2Ei) or at maximum peak (Fig. 2Eii) were observed in the nonresponding afferents. These data demonstrate that CCDC causes mechanical hypersensitivity of a sub-population of bladder afferents (Fig. 2F). www.nature.com/scientificreports/ Gpbar1 −/− mice, the mechanical hypersensitivity to distension following CCDC compared to saline was completely abolished (Fig. 3A). As in wildtype mice there was no change in muscle compliance between CCDC and saline experiments (Fig. 3B), confirming that the absence of altered sensitivity was not caused by changes in bladder muscle function. Interestingly, single unit analysis of the afferents showed that while the majority of afferents (82.9%; 29/35) reflected the overall lack of sensitivity to CCDC, mechanical hypersensitivity was observed in 17.1% (6/35) of mechanosensitive afferent fibres (Fig. 3C). These CCDC-responsive neurons demonstrated mechanical hypersensitivity specifically at 10 mmHg (Fig. 3Di) and an increase in the maximum peak  www.nature.com/scientificreports/ of afferent firing (Fig. 3Dii). In line with the overall bladder afferent data, the vast majority of mechanosensitive single units (82.9%; 29/35) showed no difference in mechanosensitivity following CCDC instillation compared to saline (Fig. 3Ei-ii).

In vivo intravesical CCDC in wild-type mice increases the number of activated dorsal horn neurons to bladder distension, an effect not apparent in Gpbar1 −/− mice.
Our results thus far have established a role for TGR5 agonists in peripheral bladder afferent hypersensitivity. To determine whether this mechanical hypersensitivity is conveyed to the central nervous system, we assessed dorsal horn neuronal activation in response to in vivo bladder distension at 40 mmHg induced pERK-immunoreactivity in lumbosacral (LS) dorsal horn neurons (Fig. 4A). We observed LS dorsal horn pERK-immunoreactive (-IR) neurons in response to saline bladder distension at 40 mmHg in wild-type mice (Fig. 4Ai). The pERK-IR neurons were identified predominantly in the regions of the dorsal grey commissure and the superficial dorsal horn, with some pERK-IR neurons in the lateral spinal nuclei and the sacral parasympathetic nucleus in sacral spinal segments (Fig. 4Aii). Following intra-bladder CCDC application we observed a significant increase in the number of pERK-IR neurons within LS dorsal horn which were distributed throughout the same regions of the dorsal horn as seen in the saline experiments (Fig. 4Ai, Aiii). As these regions are known to play a role in bladder afferent input, integration, and projection of autonomic and nociceptive visceral signalling 36,47 , an increase in pERK-IR neurons indicates an increase in central sensitivity to bladder distension and hence projection of the peripheral mechanosensitivity observed ex vivo in wild-type mice. LS spinal dorsal horn neuronal pERK-IR induced by in vivo bladder distensions following intravesical incubation with CCDC or saline was also determined in Gpbar1 −/− mice. The CCDC-induced enhanced spinal dorsal horn neuronal response to bladder distension was abolished in the Gpbar1 −/− model, with no difference in the number of LS dorsal horn pERK-IR neurons between CCDC and saline in these mice (Fig. 4Bi). The pERK-IR neurons activated by bladder distension following intravesical instillation of both saline and CCDC were observed in the same dorsal horn regions as seen in the wildtype, suggesting no differences in spinal bladder afferent signalling pathways in the Gpbar1 −/− mice (Fig. 4Bii,Biii). Taken together, these results demonstrate that activation of the TGR5 receptor is able to mediate an increase in peripheral and spinal sensitivity to bladder distension indicative of sensory neuron activation.

In vitro application of CCDC to bladder-innervating DRG neurons increases intracellular Ca 2+ .
To gain insights into the mechanism of action of neuronal TGR5 in mediating bladder afferent sensitivity, we performed live cell calcium imaging in LS DRG neurons isolated from retrogradely bladder-traced mice. Following direct application of CCDC to the neurons, a significant increase in intracellular Ca 2+ , indicated by an increase in the Fura-2 fluorescence ratio (Fig. 6Ai,Aii), was observed in 58.82% (40/68) of bladder-traced DRG neurons (Fig. 6Bi). Interestingly, the percentage of bladder-traced neurons responding to CCDC was substantially higher than the percentage on non-traced neurons, of which only 24.65% (126/511) responded (Fig. 6Bi). The average magnitude of CCDC-induced increases in intracellular Ca 2+ was also greater in bladder-traced compared to non-traced neurons, as indicated by a significantly higher maximum peak value of Fura-2 fluorescence and area under the curve of response (Fig. 6Bii,Biii). These results demonstrate the TGR5 agonist CCDC is able to cause direct activation of LS DRG neurons and indicate the possibility that bladder-innervating DRG neurons are tuned to evoke greater responses to TGR5 agonists.

In vitro CCDC-induced increases in intracellular Ca 2+ in bladder-innervating DRG neurons are partially mediated by TRPV1.
Coupling between TGR5 and TRPA1 has been previously demonstrated in cutaneous 24 and colon 26 innervating DRG neurons. Here our single-cell mRNA expression data from bladder-innervating DRG neurons demonstrated 100% co-expression of trpv1 with Gpbar1 compared to only 30% co-expression of trpa1 with Gpbar1 (Fig. 5C), suggesting a distinct intracellular signalling pathway in bladderinnervating DRG neurons. To functionally explore potential coupling between TGR5 and TRP channels in bladder afferent DRG neurons in vitro, we determined co-activation of TGR5, TRPA1, and TRPV1 in individual neurons with successive administration of CCDC, AITC (TRPA1 agonist), and capsaicin (TRPV1 agonist) using live cell calcium imaging (Fig. 7A,B). In line with our single-cell co-expression data, 87.5% (21/24) of bladder   www.nature.com/scientificreports/ traced neurons that responded to CCDC also responded to capsaicin, whilst 33.3% (8/24) of CCDC-responsive bladder-traced neurons also responded to AITC (Fig. 7B). Only 16.7% (4/24) of CCDC-responsive bladdertraced neurons were unresponsive to both AITC and capsaicin (Fig. 7B).

Pressure (mmHg) Afferent firing (imp/s)
To further explore the potential functional relationships between TGR5 and TRP channels TRPV1 and TRPA1, we measured bladder-traced DRG neuronal responses to CCDC in vitro using trpv1 −/− and trpa1 −/− mice. The percentage of bladder-traced DRG neurons responding to CCDC was reduced by approximately 50% in trpv1 −/− mice (27.3%) compared to wildtype mice (58.8%), with no obvious change observed in the trpa1 −/− mice (60.9%) compared to wildtype mice (Fig. 7C). The profile and magnitude of CCDC responses between wildtype and trpv1 −/− and trpa1 −/− mice was also examined (Fig. 7Di-iii). Whilst the few trpv1 −/− neurons that responded to CCDC show a trend towards reduced response amplitudes, there was no statistical differences in the average maximum peak of Fura-2 fluorescence (Fig. 7Dii) and area under the curve (Fig. 7Diii) of the CCDC responses. Overall, these data implicate an interaction between TRPV1 and TGR5 in subsets of bladder-innervating DRG neurons. However, as CCDC responses are also observed in some trpv1 −/− DRG neurons, this suggests interactions with other channels also occur.

In vitro CCDC-induced increases in intracellular Ca 2+ in bladder-innervating DRG neurons remain in Gpbar1 −/− mice.
To confirm the in-vitro effects of CCDC on isolated bladder-innervating DRG neurons were due to activation of the TGR5 receptor we examined CCDC responses firstly in cells isolated from Gpbar1 −/− mice and subsequently in Na V 1.8Cre-Gpbar1 −/− mice. To our surprise, responses to CCDC remained in neurons from these mice (Fig. 8). The percentage of cells responding to CCDC from Gpbar1 −/− and Na V 1.8Cre-Gpbar1 −/− mice was roughly equivalent to that from wild-type mice (Fig. 8Ai-iii,Bi-ii). The total fluorescent response to CCDC, depicted as the area under the response curve (AUC) was significantly reduced in cells from Na V 1.8Cre-Gpbar1 −/− (neuronal knockout of Gpbar1) but not Gpbar1 −/− (global knockout of Gpbar1) mice (Fig. 8Aiii,C), however, a robust response still remained. No significant differences in the maximum peak of Fura-2 fluorescence in response to CCDC were observed in cells from wildtype, Gpbar1 −/− or Na V 1.8Cre-Gpbar1 −/− mice (Fig. 8D).

CCDC-evokes increases in intracellular Ca 2+ in a small proportion of non-neuronal cells.
We have shown that the effects of CCDC in the intact bladder, namely mechanical hypersensitivity of pelvic afferents and spinal cord activation, is prevented in Gpbar1 −/− mice. Therefore, we investigated if the in-vitro effects we observed with CCDC in neurons from Na V 1.8Cre-Gpbar1 −/− and Gpbar1 −/− mice resulted from additional cell types responding to CCDC that are not present in the bladder (Fig. 9). We observed that a small population (3%) of non-neuronal cells from wildtype mice responded to CCDC (Fig. 9ABi-iii). Surprisingly, non-neuronal responses to CCDC were retained in cells from Gpbar1 −/− and Na V 1.8Cre-Gpbar1 −/− mice (Fig. 9Bi). Furthermore, the area under the response curve (AUC, Fig. 9Bii) and the maximum peak of Fura-2 fluorescence in response to CCDC (Fig. 9Biii) were not significantly reduced in cells from Gpbar1 −/− nor Na V 1.8Cre-Gpbar1 −/− mice.

Discussion
The overlapping symptoms of OAB and IC/BPS, including frequency, urgency and discomfort indicate an increase in the sensitivity of bladder afferent neurons to bladder filling. Identifying the receptors and channels involved in bladder afferent neuronal activation, and the impact these have on behavioural responses in states of health and hypersensitivity, is a crucial step to developing efficacious treatment options for patients suffering bladder hypersensitivity disorders. Here, we have identified a role for the bile acid receptor TGR5 in mediating bladder afferent mechanical hypersensitivity in vivo and ex vivo. Intravesical administration of the progesterone metabolite Pg5α and the synthetic TGR5 agonist CCDC increased afferent sensitivity to bladder distension at both peripheral and spinal levels, responses which we show to be reduced or absent in mice lacking the gene for TGR5 (Gpbar1).
These findings are important as endogenous TGR5 agonists, including bile acids and progesterone metabolites are present at low levels in the urine of healthy subjects 33,34 and are increased dramatically under different The pressure to volume relationship during bladder distension is unchanged following CCDC administration compared to saline, indicating no change in muscle compliance in preparations from Gpbar1 −/− mice (ns, P > 0.05). (C) Whilst the majority of afferents (82.9%) reflected the overall results with no change in afferent firing (29 out of 35; non-responders), 6 out of 35 (17.1%) afferents displayed enhanced responses to distension following CCDC with an increase in mechanosensitivity. (D) In CCDC-responsive afferents, (i) a significant increase in imp/s was observed following CCDC administration overall (**P < 0.01) and specifically at a distension pressure of 10 mmHg (**P < 0.01). (ii) the maximum imp/s following CCDC administration was significantly increased compared to saline in responders (**P < 0.01). (E) In non-responding afferents, (i) no change in imp/s at any distension pressure was observed in non-responders between CCDC and saline distensions (ns, P > 0.05), whilst (ii) no significant difference was observed between the maximum imp/s of non-responders following administration of CCDC compared to saline (ns, P > 0.05). P values above are based on two-way ANOVA (A,B,Di,Ei), with subsequent Sidak's post hoc test significance at individual data points indicated on (Di), Wilcoxon matched-pairs signed rank test (Dii,Eii).

Scientific Reports
| (2022) 12:9920 | https://doi.org/10.1038/s41598-022-14195-w www.nature.com/scientificreports/ conditions, including pregnancy and liver disease 32,50 . As such, the increased sensitivity of these mechanosensitive bladder afferents following intravesical administration of Pg5α suggests an endogenous role for TGR5 agonists in mediating afferent hypersensitivity. The CCDC-induced increase in bladder afferent firing to distension largely mirrored that of PG5α, although the sensitisation was observable from earlier distension pressures following CCDC administration compared to PG5α. Importantly, the overall bladder afferent hypersensitivity induced by CCDC was abolished in Gpbar1 −/− mice and the percentage of responding afferents greatly reduced. These findings of afferent sensitisation by TGR5 agonists have physiological relevance as projections from bladder afferents terminate within the lumbosacral spinal dorsal horn 10,51 . Following peripheral activation, these neurons release neurotransmitters within the dorsal horn which act on second order neurons. Sub-populations of second order spinal neurons involved in bladder sensory pathways have been identified within the superficial dorsal horn (SDH), the dorsal grey commissure (DGC) and the sacral parasympathetic nucleus (SPN) 6,16,36,[51][52][53][54][55] . These second order neurons relay peripheral bladder sensory input to the brain, resulting in autonomic micturition reflexes and cognitive perception 6,36,47,56 . Here, we demonstrate in sacral spinal segments that following CCDC and bladder distension there is an increase in the number of pERK-IR neurons within the SDH, the DGC, the lateral spinal nuclei and the SPN. This indicates peripheral bladder afferent hypersensitivity induced www.nature.com/scientificreports/ by TGR5 agonism is relayed into the spinal cord and subsequent central pathways. These findings correlate well with our previous studies whereby intravesical application of histamine or Mrgpr agonists increases bladder afferent responses to bladder distension, resulting in increased neuronal activation within the spinal cord dorsal horn regions associated with micturition and nociception 16,17 . Our mRNA expression results confirm Gpbar1 expression in all layers of the mouse urinary bladder, building on a previous study showing expression of the TGR5 gene Gpbar1 mRNA in whole rat bladder 23 . Whilst TGR5 is known to play an important role in smooth muscle relaxation, particularly in the gallbladder and the stomach 57-59 , it is particularly important to note that in the current study bladder muscle compliance was unchanged following intravesical administration of either Pg5α or CCDC. This indicates the Pg5α-or CCDCinduced hypersensitivity of bladder afferents to distension occurs at the level of the afferent ending. Importantly   24,26 . Both studies further analysed co-expression with Trpa1 and Trpv1, which are common downstream targets for GPCR signalling and have both been shown to play a role in mediating itch and pain sensation 15,48 . These previous studies indicate that TRPA1 is the key downstream effector coupled to neuronal TGR5, with both cutaneous and colonic afferent hypersensitivity to CCDC abolished in trpa1 −/− mice 24,26 . Previous expression data largely supports this, as cutaneous-and colon-innervating DRG neurons show high co-expression with both trpa1 (100% in cutaneous and 83% in colonic Gpbar1-positive DRG neurons) and trpv1 (60% in cutaneous and 78% in colonic Gpbar1-positive DRG neurons) 24,26 . Another study assessing Gpbar1 mRNA expression in small diameter DRG neurons showed lower associations with both trpa1 (41% of Gpbar1-positive small diameter DRG neurons) and trpv1 (32% of Gpbar1-positive small diameter DRG neurons) 29 . Interestingly, we show bladder-innervating DRG neurons differ to cutaneous and colon-innervating DRG neurons, with 100% of bladder-innervating Gpbar1-expressing DRG neurons co-expressing trpv1, but only 30% co-expressing trpa1. Our in vitro functional calcium imaging data showed CCDC activated a population of bladder-innervating DRG neurons, and that the magnitude of these CCDC responses were greater in bladder-innervating neurons than non-bladder innervating neurons, suggesting an enhanced role in bladder pathways. Bladder-innervating   24,26 . However, unlike these previous studies, significant functional co-expression was observed between TGR5 and TRPV1 (CCDC and capsaicin responsive) with less interaction observed between TGR5 and TRPA1 (CCDC and AITC responsive). These data correspond with our mRNA expression data which demonstrates extensive Gpbar1 and trpv1 co-expression, suggesting an alternative downstream coupling mechanism for TGR5 to that seen in colonic or cutaneous DRG neurons. The potential associations of in vitro CCDC activity with TRPA1 and TRPV1 were further dissected using DRG from trpa1 −/− and trpv1 −/− mice. Data from these DRG neurons demonstrate that CCDC-induced activation of bladder-innervating DRG neurons was greatly reduced in trpv1 −/− DRG neurons, whilst no significant differences in CCDC responses were observed in the absence of trpa1. Together, these data indicate that TGR5 signalling in bladder afferents is partially mediated by TRPV1 as a potential downstream effector.  www.nature.com/scientificreports/ Our ex vivo and in vivo data with CCDC are relatively clear cut in the role TGR5 plays in bladder afferent hypersensitivity, with agonist responses reduced or absent in Gpbar1 −/− mice. However, the proportion of bladder-innervating DRG neurons responding to CCDC is greater than the single cell mRNA expression data predicts. Furthermore, in these calcium imaging studies CCDC responses were still observed in DRG from Gpbar1 −/− (global knockout of Gpbar1) and Na V 1.8-Gpbar1 −/− (neuronal knockout of Gpbar1 as 98% of bladderinnervating DRG neurons express Na V 1.8 37 ) mice. Whilst CCDC is a synthetic agonist developed to be TGR5specific 38 it is unclear if it can also act via additional receptors. For example, in addition to TGR5 28 progesterone metabolites can act on progesterone receptors and GABA A receptors 60,61 , although GABA A receptor agonists inhibit rather than sensitize visceral afferents 62 . In addition, recent data show that some bile acids can also act through MrgprX4 25,63 . In the current study we also found that CCDC activated a small percentage of nonneuronal cells and that these responses were similar between wildtype, Gpbar1 −/− and Na V 1.8-Gpbar1 −/− DRG neurons. A recent study has shown in human and monkey that TGR5 is highly expressed in satellite glial cells   25,63 . It is possible that some of the disparity observed in our DRG calcium imaging studies are related to indirect actions, whereby satellite glial cells release factors in response to CCDC application and that these factors then act on and activate DRG neurons. These discrepancies aside overall our mRNA expression, ex vivo and in vivo functional evidence indicate that the physiological actions of both the synthetic and the endogenous TGR5 agonists are mainly due to activation of TGR5. In particular, the enhanced dorsal horn neuronal activation in response to CCDC and bladder distension is completely lost in Gpbar1 −/− mice, suggesting TGR5 expressing bladder-innervating DRG neurons are crucial for these sensitizing actions of CCDC in vivo. How bile acid or progesterone metabolite urinary content is altered during bladder hypersensitivity is not yet known. However, an important consideration in determining any role for TGR5 in OAB and IC/BPS is the permeability of the urothelium. Increased urothelial permeability is a common finding in IC/BPS, which may allow increased access of urinary contents, such as bile acids and progesterone metabolites, to access the underlying tissue and nerve endings within the bladder wall 1,64 . Further studies are required to determine the impact of TGR5 signalling in bladder afferents during states of hypersensitivity and any changes specific to this pathway including receptor expression and agonist concentrations in the urine and tissue.
In conclusion, this study has identified the expression and function of the bile acid receptor TGR5 in bladderinnervating afferent DRG neurons. Intravesical administration of TGR5 agonists in the urinary bladder results in TGR5-dependent enhanced mechanosensitivity to bladder distension and increased spinal dorsal horn neuronal activity, indicating a role for TGR5 in mediating the sensitivity of bladder sensory signalling pathways. Further, we have shown that TGR5 activation in bladder-innervating neurons is partially mediated by TRPV1. As irritant-sensing GPCRs are now known to enhance bladder mechanosensitivity, TGR5 and its related receptors may represent potential targets for future treatment in conditions of bladder hypersensitivity.